We are requesting support to continue and extend our NMR studies of the regulation and control of metabolism in vivo. We are planning in the period of this grant to take advantage of advances we have made during the past three years, particularly those in 1H NMR, and to use the methods of 13C and 31P NMR in new studies. There are three main areas of research planned: the rat brain in vivo, the perfused rat heart and suspensions of yeast cells. In the rat brain we are planning to make our proton NMR methods more quantitative so that we can use the sensitivity and resolution of the proton spin echo difference spectra to determine meaningful concentrations of brain metabolites. Once this is done we plan to study glycolysis in the brain during steady state hypocapnia and the subsequent transient changes after ischemia. We also intend to study amino acid pools in the brain, particularly glutamate and glutamine in conjunction with Gamma-aminobutyric acid (GABA) in order to study metabolic compartmentation of these compounds. These will be monitored by proton observe - 13C decoupled experiments after the infusion of 13C labeled substrates. Effects of different states of the brain upon these fluxes will be evaluated, and it is expected that the label distribution will give us information about the biochemical pathways. In the perfused heart we will concentrate on two problems: first we will evaluate the glycolytic flux and see whether by determining the allosteric effectors of PFK we can mimic the in vivo fluxes in an in vitro experiment; second we will evaluate phosphorylase activity by measuring the rate of formation of lactate during ischemia and seeing the effects of an inhibitor of phosphorylase b (2 deoxyglucose-6-phosphate) upon this rate. In the year we are planning to study by saturation transfer, the exchange reactions responsible for the Pi consumption activity. Thorough inhibitor studies will be employed to resolve possible contributions from the mitochondrial ATPase, the plasma membrane ATPase, and glyceraldehyde 3-phosphate dehydrogenase/phosphoglycerate kinase coupled reaction. Once the contributions to saturation transfer of these individual exchange reactions have been identified, S.T. experiment will be conducted under various conditions to obtain kinetic information about yeast cellular metabolism.